Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometric Comparison ofN-Acetyl- andN-Pivaloyl Amino Acid Esters to Measure15N Isotopic Abundances in Physiological Samples: A Pilot Study on Amino Acid Synthesis in the Upper Gastro-intestinal Tract of Minipigs

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Abstract
There is evidence that in animals and also in humans, non-specific nitrogen is used for de novo synthesis of indispensable amino acids by the microflora of the gastro-intestinal tract. Gas chromatography/combustion/isotope ratio mass spectrometry was applied to investigate whether lysine of intestinal origin is available for body protein synthesis. Two minipigs with an end-to-end ileorectal anastomosis received orally equimolar amounts of 15N as ammonium chloride or urea twice a day for 10 days. Samples of blood and ileal digesta were collected before and throughout the 10 days; tissue samples were taken at termination of the experiment. The N-acetyl-n-propyl (NAP)- and N-pivaloyl-i-propyl (NPP)-amino acid esters were evaluated for the determination of the 15N content of lysine and 16 other amino acids ranging from natural abundance to an enrichment of 0.6 APE 15N in a complex mixture of proteinogenic amino acids and several matrices. At natural abundances for all amino acids analysed, NAP and NPP derivatives gave mean precisions of 0.5 and 0.3/1000 delta 15N, respectively. The mean precision for NPP derivatives at enrichments between 0.42 and 1.10 AP 15N ranged between 1.0-15.0/1000 delta 15N. 15N from ammonium chloride was incorporated into lysine and in all other amino acids of serum albumin to a 2.5 times higher degree than from urea. Somewhat lower, but significant, lysine enrichments were detected in liver, duodenum and jejunum. After 10 days of ingestion of 15N-labeled urea a significant 15N enrichment in lysine of serum albumin could not be detected, although lysine in the ileal digesta was significantly labeled by day 5. This is the first report providing evidence that the microflora in the upper gastrointestinal tract of pigs is capable of synthesizing lysine de novo and that this lysine is available for body protein synthesis.

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